kinases - introduction
DESCRIPTION
Structural Insights into Kinase Inhibition Ramesh Sistla and Subramanya H.S. Aurigene Discovery Technologies Ltd. #39-40, KIADB Industrial Area, Electronic City Phase II Bangalore 560 100. Kinases - Introduction. Kinases are enzymes that catalyze phosphorylation - PowerPoint PPT PresentationTRANSCRIPT
Structural Insights into Kinase InhibitionRamesh Sistla and Subramanya H.S.
Aurigene Discovery Technologies Ltd.#39-40, KIADB Industrial Area, Electronic City Phase II
Bangalore 560 100
AURIGENE……Acccelerating Discovery 2
Kinases - Introduction• Kinases are enzymes that
catalyze phosphorylation• ATP + protein = ADP + phosphoprotein
• Key signaling enzyme• Human genome encodes > 500
kinases - Kinome• They have been implicated in
different diseases including cancer, metabolic disorders and central nervous system indications.
• Depending on the amino acid a kinase phosphorylates, they are known as Serine/Threonine or Tyorsine kinases. www.cellsignal.com
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Signaling Cascades• The figure shows the
involvement of kinases in cell proliferation and survival.
• In this cascade the phosphorylation of each kinase by its upstream kinase serves as a signal for downstream activity.
• Inhibiting the pathway through inhibition of kinase involved in the pathway is an attractive proposition
Current Medicinal Chemistry, 2008 Vol. 15, No. 29 3037
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Promise of Kinase InhibitorsDruggable Genome
• Kinases are an attractive target class– Druggability– Early successes (FDA approval of some of the kinase inhibitors)
• Possibility of structure guided design – Large number of crystal structures in complex with inhibitors are available
Structure Identifier Target
AMN-107 BCR/ABL
STI-571 BCR/ABL
BMS-354825
BCR/ABL+
OSI-774 Erbb
O
NH
F
FF
N CH3NCH3
NH
N
N
N
O
NH
CH3
NH
N
N
N
N
NCH3
O N
NH
N
CH3
N
N
NOH
SNH
CH3
Cl
CH3OO
NN
NHCH
OOCH3
Imatinib
Dasatinib
Some Advanced Kinase Inhibitors
Kinome
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General Structure of Kinases• Bi-lobial structure• N-termial lobe
– Mainly made of beta-sheets and connecting loops
– One functionally important helix
• Both lobes joined by a loop called hinge.
• ATP binding pocket is in the interface between the lobes
• C-terminal lobe– Mainly made of α-helices
• Activation loop spans both N- and C-terminal lobes
C-terminal lobe
N-terminal lobe
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Important Structural Elements
• Glycine rich loop– Closes in on the ATP
• Helix C– Plays an important role in catalysis
• Hinge– Adenosine moiety of the ATP makes bidentate
H-bond with this region
• Activation loop– Starts with conserved sequence DFG and ends
with APE.
…GxGxxG…
Helix-C
DFG……APE loopHinge ATP
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Binding of ATP and Catalysis
γ-phosphate coordinates with the metal
•Activation loop (DFG……APE) provides docking site for the substrate•Highly disordered and usually unresolved in the x-ray structures
Orientation of the DFG motif critical for the phosphorylation
HingeMetal
Metal
SubstratePhosphate
H-bonds
S T
Y
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Important Residues
• In the active conformation of the kinases, a conserved Lys residue makes a salt bridge with a conserved Glu residue in the middle of the helix-C.
• This interaction ensures the positioning of the amino acid Asp (of the DFG motif) to coordinate with the γ-phosphate, the divalent metal ion and catalytic water molecule to facilitate catalysis
N-terminal lobe
C-terminal lobe
ATP
Lys
GluAsp Water
Metal
Helix-C
Close up of the catalytic machinery
Salt bridge
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Kinase Inhibitors• In most cases, inhibitors compete with ATP in order to inhibit the kinase
– Such inhibitors are ATP mimetics in the sense that they make interactions similar to what ATP makes.
Hinge
G-loop
ATPInhibitor ATP
Inhibitor
Ribose pocketPhosphate pocket
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Various Subsites in Kinases
An example of a kinase inhibitor bound in the ATP pocket is shown.
Apart from hinge region interaction and solvent interaction, the inhibitor occupies a deeper hydrophobic cavity, also known as selectivity pocket
Size of an amino acid preceding the hinge region controls the accessibility to the deeper pocket – Gatekeeper, (Typically Met/Leu/Thr/Ile/Tyr)
Hinge
ATP
Inhibitor
Deeper cavity
Solvent
Gatekeeper
PDB: 2C6E/1MQ4
Hinge
ATP
Inhibitor
Deeper cavity
Solvent
Gatekeeper
PDB: 2C6E/1MQ4
Schematic of the binding pockets
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Type I Inhibitor- Dasatinib
1nM10nM100nM1μM10μM
• Dasatinib was developed as a c-Src/BCR-Abl inhibitor but was found to hit many other kinases.
• Cross reactivity mainly within the TK family; Approved by FDA
Ref: Karaman et. al., NATURE BIOTECHNOLOGY VOLUME 26 NUMBER 1 JANUARY 2008
Hinge
Solvent
Deeper pocket
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DFG-IN vs DFG-OUT
Gly rich loopHelix-C
DFG-In
DFG-Out
• The activation loop (DFG….APE) has to be IN when the kinase is active – DFG “in” conformation• The DFG loop has been shown to be in an “out” position when kinases are inactive. • This can be exploited in the design of inhibitors.
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DFG-IN vs DFG-OUT• Differences between
DFG IN and DFG OUT structures are exemplified.
• DFG loop in OUT position will clash with phosphate of ATP
• When DFG moves to OUT helix-C also moves away creating the pocket shown by bold red arrow.
• Gleevec binds to the DFG-OUT conformation of the C-Abl kinase.
ATPGleevec
DFG IN
DFG OUT
Helix-C
PDB:1T46
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Example of Type-II Inhbition
Phe-out conformation
BIRB-796 binds to p-38 in the Phe-out conformation
Ref: Karaman et. al., NATURE BIOTECHNOLOGY VOLUME 26 NUMBER 1 JANUARY 2008
• The doublet of H-bonds with E-111 (helix-C) and D-207 (DFG loop) backbone is very important• Hence a urea or amide is the common feature in these inhibitors
PDB:1KV1
Hinge
Schematic of the binding pockets
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Some Known DFG OUT Inhibitors
2ofvLck – DFG out
2og8Lck – DFG out
Bioorg.Med.Chem.Lett. 17: 2886-2889
2oo8Tie – DFG out
2p4iTie – DFG out
J.Med.Chem. 50: 611-626
2oscTie – DFG out
Bioorg.Med.Chem.Lett. 17: 2886-2889 J.Med.Chem. 50: 611-626
2p2iKDR – DFG out
Apart from a hinge binding group, the common feature in these molecules is existence of the bi-aryl amide/urea group which makes interaction with Glu (helix-C) and Asp (DFG loop)
N H
O
R
R
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Allosteric Kinase Inhibition – Type III• Certain kinases have an
allosteric pocket in which an inhibitor can co-bind with ATP
• The phosphorylation of the substrate is prevented by unavailability of the catalytic Asp
• There are no hinge region interactions in these inhibitors.
DFG loop
Helix-C
ATP
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A Still Different Type of Inhibitor?• Recently Merck published the
co-crystal structure of CHK1 kinase with an inhibitor that is bounds far away from the active site.
• DFG loop is has IN conformation, but the inhibitor probably occupies substrate binding site.
• Such inhibitors are not being designed yet. They could be results of HTS campaigns.
PDB:3F9N
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SBDD at Aurigene
All the structural biology efforts are to aid in more focused medicinal chemistry